U.S. patent application Ser. No. 10/059,993 entitled Multi-Channel Bio-Separation Cartridge, concurrently filed on Jan. 28, 2002, which is assigned to BioCal Technology, Inc., the assignee of the present invention, and which is fully incorporated by reference herein.
1. Field of the Invention
The present invention relates to detection techniques in bio-analysis, particularly optical detection in a multi-channel bio-separation system, and more particularly detection of emissions from radiation excitations in multi-channel capillary based electrophoresis. The present invention further relates to bio-separation instrument incorporating the detection scheme of the present invention.
2. Description of Related Art
Bioanalysis, such as DNA analysis, is rapidly making the transition from a purely scientific quest for accuracy to a routine procedure with increased, proven dependability. Medical researchers, pharmacologists, and forensic investigators all use DNA analysis in the pursuit of their tasks. Yet due to the complexity of the equipment that detects and measures DNA samples and the difficulty in preparing the samples, the existing DNA analysis procedures are often time-consuming and expensive. It is therefore desirable to reduce the size, number of parts, and cost of equipment, to make easy sample handling during the process, and in general, to have a simplified, low cost, high sensitivity detector.
One type of DNA analysis instruments separates DNA molecules by relying on electrophoresis. Electrophoresis techniques could be used to separate fragments of DNA for genotyping applications, including human identity testing, expression analysis, pathogen detection, mutation detection, and pharmacogenetics studies. The term electrophoresis refers to the movement of a charged molecule under the influence of an electric field. Electrophoresis can be used to separate molecules that have equivalent charge-to-mass ratios but different masses. DNA fragments are one example of such molecules.
There are a variety of commercially available instruments applying electrophoresis to analyze DNA samples. One such type is a multi-lane slab gel electrophoresis instrument, which as the name suggests, uses a slab of gel on which DNA samples are placed. Electric charges are applied across the gel slab, which cause the DNA sample to be separated into DNA fragments of different masses.
Another type of electrophoresis instruments is the capillary electrophoresis (CE) instrument. CE refers to a family of related analytical techniques that uses very strong electric fields to separate molecules within narrow-bore capillaries (typically 20-100 um internal diameter). CE techniques are employed in seemingly limitless applications in both industry and academia. Gel- and polymer network-based CE has revolutionized studies of nucleic acids; applications include DNA sequencing, nucleotide quantification, and mutation/polymorphism analysis. By applying electrophoresis in a fused silica capillary column carrying a buffer solution, the sample size requirement is significantly smaller and the speed of separation and resolution can be increased multiple times compared to the slab gel-electrophoresis method. These DNA fragments in CE are often detected by directing light through the capillary wall, at the components separating from the sample that has been tagged with a fluorescence material, and detecting the fluorescence emissions induced by the incident light. The intensities of the emission are representative of the concentration, amount and/or size of the components of the sample.
Some of the challenges in designing CE-based instruments and CE analysis protocols relates to sample detection techniques. In the case of fluorescence detection, considerable design considerations had been given to, for example, radiation source, optical detection, sensitivity and reliability of the detection, cost and reliability of the structure of the detection optics.
CE with the use of the fluorescence method provides high detection sensitivity for DNA analysis. Fluorescence detection is often the detection method of choice in the fields of genomics and proteomics because of its outstanding sensitivity compared to other detection methods. Two prevailing fluorescence detection modes are confocal scanning laser induced fluorescence (LIF) and sheath flow detectors.
The main drawback of the sheath flow detector is the highly sophisticated flow system needed to ensure a reliable sheath flow. Extreme demands are put on the optical and mechanical component tolerances in order to meet the robustness demands of end-users. The sensitivity of the device is very good, but it is not obvious that this principle of fluorescence detection is suited for a high-throughput yet low cost DNA analysis. The scanning confocal detector is based on scanning the optical system. The use of moving parts is not ideal when considering simplicity, robustness and lower cost of the instrument. Also, the shallow focal depth of the microscope objective puts severe demands on the mechanical and optical component tolerances. Further, the optical scanning principle reduces the duty cycle per capillary, which may impair the sensitivity when scaling up the instrument further for very high-throughput purposes.
One of the most expensive hardware components for many commercially available CE instruments with LIF detector is typically a fluorescence excitation light source, which can be a gas discharge lamp (mercury or xenon) or a laser (gas, solid state with second harmonic generation, dye, or semiconductor), that are bulky, expensive, inefficient and difficult to couple one""s light output into optical fibers, thus preventing miniaturization of the optical detection system. These light sources hinder development of small size, high-throughput and cost-effective analytical instruments, with the convenience required for rapid detection.
Separation of DNA fragments by size using gel-filled capillaries has advantages over the classical slab gel-based separations in terms of speed and resolution. However, most commercially available CE instrumentation has only one capillary, and only one sample may be analyzed at a time. In a commercial automated DNA sequencer utilizing a slab gel system (such as the Applied BioSystems 377 instrument), 36 samples may be analyzed simultaneously. In order for capillary electrophoreis based DNA fragment analyzing instrumentation to be competitive on a throughput basis, it has been necessary to develop instruments that can run more than one sample at a time. The principle involved in DNA fragment Analyzing instruments (such as PACE 5000 LIF, PACE MDQ LIF of Beckman Coulter) using a single-capillary instrument can be extended to multi-capillary system. However, the design of a multi-capillary DNA Analyzer utilizing LIF based detection optical systems (i.e. 8-capillary CEQTM 2000XL Instrument of Beckman Coulter) are considerably more costly and complicated than the classical slab gel-based systems. Other examples of commercially available multi-capillary CE instruments include instruments developed by ABI, SpectruMedix and Pharmecia, which shared similar drawbacks.
Another fluorescence detection method illuminates the interiors of multiple capillaries simultaneously, and collects the light emitted from them. As in U.S. Pat. No. 5,790,727, the capillaries in a parallel array form an optical wave guide wherein refraction at the cylindrical surfaces confines illuminating light directed in the plane of the array to the core of each adjacent capillary in the array. However, because only one light source is used for the illumination, there is cross talk between the different separation channels defined by the capillaries. Due to the existence of scatter light, cross talk cannot be prevented and the contrast ratio of detected signals will be poor due to noise in the fluorescence emission. Furthermore, prior art single illumination source for multiple channels makes multi-wavelength LIF detection more complicated.
It is therefore desirable to develop a low-cost high throughput multi-channel detection scheme for bio-separation, which would overcome the limitations in the prior art.
The present invention provides a simplified, low cost, efficient, highly sensitive, and high throughput multi-channel detection configuration for bio-separation (e.g., CE), which overcomes the drawback of the prior art. The present invention provides a multi-channel detection scheme based on a multi-radiation source/common detector configuration, in which detection is conducted in a time-staggered, and/or time-multiplexed detection for the channels. A single detector is coupled to a plurality of radiation sources, in a one detector/many radiation sources configuration. Each radiation source directs radiation at one detection zone of a single separation channel, and a single detector is applied to detect light emissions from the detection zones of several separation channels. There may be more than one detector in the entire detection system, each serving multiple radiation sources.
Bio-separation may be conducted simultaneously in all the channels in parallel, with detection time-staggered and/or time-multiplexed with respect to the light sources. The light sources direct radiation at the detection zones in a predetermined sequence in a cyclic manner, with the detector output synchronized to the light sources by a controller. The radiation sources and the detector are pulsed in synchronization in a time-multiplexed manner. The controller controls the detector and radiation sources in a manner to effect detection of radiation emissions from the multiple separation channels in predetermined detection cycles, wherein each detection cycle is repeated at a frequency to provide a desired detection time or duration. The controller controls the radiation sources and detector in a manner to effect detection in a repeated scanning manner, across the detection zones of the separation channels, in a time-staggered type detection. In accordance with the present invention, cross talk between channels is virtually eliminated. No moving parts are required for directing the light source or detection.
In one embodiment, low cost light emitting diodes (LEDs) (e.g., super bright LEDs) or laser diodes, instead of expensive high power lasers may be used as light sources. Alternatively, low energy lasers may be used. The incident light from the light sources may be separately directed to the detection multi-channel detection zones using optic fibers. The emitted light from the multi-channel detection zones may be directed to one or more common detectors using optic fibers. Since no moving parts are necessary, component count can be reduced. By eliminating or reducing cross talk between channels, the optical detection system design can be much simplified. Accordingly, the cost, reliability, and ease of use of the instrument is improved.
In a particular embodiment of the present invention, the detection scheme of the present invention is configured for radiation induced fluorescence detection in a multi-channel bio-separation instrument. In another embodiment, the detection scheme is configured for radiation induced fluorescence detection in a capillary electrophoresis instrument.
In another aspect of the present invention, the present invention provides a bio-separation instrument that incorporates the detection scheme of the present invention.